Cell-Free Translation System

Abstract

The present invention relates to a new cell-free translation system. In particular, the invention relates to a cell-free reaction system for translating in vitro a RNA into a protein, said reaction system comprising a ribosome-depleted red blood cell lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. The invention also pertains to a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest using the cell-free reaction system of the invention. The invention also relates to the use of (i) a ribosome-depleted red blood cell lysate, and (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes, for producing a cell-free translation system.

1 . A method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest, the method using a translation reaction mixture comprising: (i) a ribosome-depleted red blood cells lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. 
     
     
         2 . The method according to  claim 1 , said method comprising:
 pre-a) optionally preparing a translation reaction mixture comprising:
 (i) a ribosome-depleted red blood cells lysate, 
 (ii) ribosomes isolated from eukaryotic cells, 
 with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes; 
   a) to form a translation system, contacting the translation reaction mixture as defined in step (pre-a) with:
 (i) the ribonucleic acid template (RNA); or 
 (ii) a transcription reaction mixture comprising
 (i) a DNA encoding the amino acid sequence of interest, and 
 (ii) the necessary element for transcribing said DNA into the ribonucleic acid template; 
 
   and   b) incubating the translation system of (a) for a time sufficient to achieve translation of the ribonucleic acid template into the amino acid sequence of interest.   
     
     
         3 . The method according to  claim 1 , said method comprising:
 pre-a) transfecting a DNA encoding the amino acid sequence of interest into eukaryotic cells, with proviso that the eukaryotic cells are not reticulocytes;   a) after a time sufficient to allow the eukaryotic transfected cells to achieve RNA transcription and to begin translation, isolating ribosomes from the transfected cells of step (pre-a) under conditions that allow the RNA produced from the DNA encoding the amino acid sequence of interest to be isolated together with the ribosomes; and   b) incubating a translation system comprising (i) a ribosome-depleted red blood cells lysate, (ii) ribosomes isolated from the eukaryotic cells in step (a) for a time sufficient to achieve translation of the ribonucleic acid template into the amino acid sequence of interest.   
     
     
         4 . The method according to  claim 2 , wherein step (b) is conducted during at least 15 minutes. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . A cell-free translation reaction system for translating in vitro a RNA into a protein comprising: (i) a ribosome-depleted reticulocyte lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. 
     
     
         8 . An in vitro method for analyzing an actively translated transcriptome of eukaryotic cells of interest comprising:
 a) isolating ribosomes from eukaryotic cells of interest under conditions that allow RNAs produced in the eukaryotic cells of interest to be isolated together with the ribosomes;   b) incubating a translation system comprising (i) a ribosome-depleted red blood cells lysate and (ii) ribosomes and RNAs isolated from the eukaryotic cells of interest in step a), for a time sufficient to achieve translation of the RNAs, isolated with the ribosomes in step a), into the corresponding amino acid sequences,   
       with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells of interest from which ribosomes are isolated are not nuclease rabbit untreated reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells of interest from which ribosomes are isolated are not nuclease treated rabbit reticulocytes; and
 c) identifying and optionally quantifying the amino acid sequences obtained in step b). 
 
     
     
         9 . A kit for translating in vitro a ribonucleic acid template into an amino acid sequence of interest comprising, in separate containers or in the same container, (i) a ribosome-depleted reticulocyte lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. 
     
     
         10 . A kit for producing a translation reaction mixture comprising, in separate containers or in the same container, (i) a ribosome-depleted reticulocyte lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. 
     
     
         11 . A kit according to  claim 9 , wherein said kit further comprises, in separate containers or in the same container, at least one element chosen from the group consisting of:
 at least a tRNA;   at least an aminoacyl-tRNA synthetase;   at least an initiation factor;   at least an elongation factors;   at least a termination factor;   at least a chaperone;   at least a foldase;   at least an amino acid;   at least a labelled amino acid, for instance a radiolabelled amino acid;   at least an energy source;   at least an energy regenerating system;   salts;   a buffer solution.   
     
     
         12 . A kit for analyzing an actively translated transcriptome of eukaryotic cells of interest comprising, in separate containers or in the same container:
 a ribosome-depleted reticulocyte lysate, and   at least one element chosen from the group consisting of:
 at least a labelled amino acid, 
 at least a biotinylated puromycin, and 
 at least an immobilized streptavidin. 
   
     
     
         13 . The method according to  claim 1 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are not reticulocytes. 
     
     
         14 . The method according to  claim 1 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit red blood cells. 
     
     
         15 . The method according to  claim 1 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         16 . The method according to  claim 1 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from a red blood cell lysate treated with a nuclease. 
     
     
         17 . The method according to  claim 1 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit reticulocytes treated with a nuclease, and the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         18 - 22 . (canceled) 
     
     
         23 . The kit according to  claim 10 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are not reticulocytes. 
     
     
         24 . The kit according to  claim 10 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit red blood cells. 
     
     
         25 . The kit according to  claim 10 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         26 . The kit according to  claim 10 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from a red blood cell lysate treated with a nuclease. 
     
     
         27 . The kit according to  claim 10 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit reticulocytes treated with a nuclease, and the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         28 . A method for in vitro producing a peptide, comprising the use of a translation reaction mixture comprising (i) a ribosome-depleted red blood cell lysate, (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. 
     
     
         29 . A method for producing translation reaction mixture comprising the use of a ribosome-depleted reticulocyte lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. 
     
     
         30 . The method according to  claim 3 , wherein step (b) is conducted during at least 15 minutes. 
     
     
         31 . The method according to  claim 2 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are not reticulocytes. 
     
     
         32 . The method according to  claim 3 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are not reticulocytes. 
     
     
         33 . The method according to  claim 2 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit red blood cells. 
     
     
         34 . The method according to  claim 3 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit red blood cells. 
     
     
         35 . The method according to  claim 2 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         36 . The method according to  claim 3 , wherein the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         37 . The method according to  claim 2 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from a red blood cell lysate treated with a nuclease. 
     
     
         38 . The method according to  claim 3 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from a red blood cell lysate treated with a nuclease. 
     
     
         39 . The method according to  claim 2 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit reticulocytes treated with a nuclease, and the eukaryotic cells from which ribosomes (ii) are isolated are human cells. 
     
     
         40 . The method according to  claim 3 , wherein the ribosome-depleted red blood cell lysate (i) is obtained from rabbit reticulocytes treated with a nuclease, and the eukaryotic cells from which ribosomes (ii) are isolated are human cells.